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Mitigated suppressive function of regulatory T cells (Treg) upon Th17-inducing cytokines in oligo- and polyarticular Juvenile Idiopathic Arthritis (JIA) patients

Background The plasticity of T helper-17 (Th17) and regulatory T (Treg) cells may be a clue to pathogenesis of Juvenile Idiopathic Arthritis (JIA). It is still unclear, whether targeted suppression of Interleukin (IL)-17 is able to influence regulatory function of Treg to control pro-inflammatory effectors in JIA. This study aimed to assess the effect of a Th17-stimulating cytokine environment and of IL-17A-inhibition on phenotype plasticity and suppressive function of Treg derived from JIA patients. Methods Th17 and Treg characteristics of CD4+ helper T cells were investigated in blood samples of JIA patients with oligo- and polyarticular pattern and healthy controls (HC). Isolated CD4+CD25+CD127− cells defined as Treg were cultivated with Th17-inducing cytokine environment as well as with IL-17A-inhibitors and analyzed for plasticity of phenotype by flow cytometry. Furthermore, inhibitory function of Treg on autologous effectors after cultivation with these stimuli was determined by suppression assays. Results Our findings demonstrated significantly elevated proportions of Th17 and Th17-like Treg in JIA compared to HC. After incubation with Th17-inducing stimuli, increased FoxP3 expression in separated Treg in JIA and an impaired suppressive capacity in JIA and HC were found. Blockade of IL-17A resulted in adjustment of FoxP3-expression in JIA to proportions found in controls and in regular suppressive function. Conclusions Our results demonstrate an induction of FoxP3 expressing Treg by Th17-inducing cytokines with concomitant mitigated suppressive function. In contrast, specific IL-17A blockade maintains suppressive Treg function and adjusted FoxP3-expression in JIA to levels found in controls. These findings may help to provide experimental evidence for the successful clinical use of IL-17A inhibition in JIA patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12969-022-00680-z.

Background
T cells play a central role in the pathogenesis of Juvenile Idiopathic Arthritis (JIA) with activated T cells accumulating in inflamed joints [1]. Research has been focusing on the balance of Th17 cells and regulatory T cells (Treg) with so far inconclusive results. Both subpopulations were elevated in the peripheral blood of children with active JIA [2]. Other studies found lower Treg frequencies in the peripheral blood of JIA compared to healthy individuals and a reciprocal relationship between Th17 and Treg cells as well as of specific transcription factors RORγt and FoxP3, respectively, in synovial fluid of inflamed joints [3][4][5]. Treg themselves are usually divided into naturally occurring/thymic and induced/peripheral Treg with the latter developing out of naive T cells by gaining FoxP3 expression via CD28costimulation and stimulation with IL-2 and TGFβ [6].
FoxP3 as one of the most important Treg markers is discussed to reflect stability and suppressive function of Treg [7,6]. Contentious findings show an unstable FoxP3-expression in inflammatory environments driving Treg to convert into proinflammatory cells [8,9] whereas others showed a stable FoxP3-lineage of Treg even in infectious and autoimmune environments [10]. Moreover, FoxP3 transcription is persistently increased during inflammation which is proposed as a possible mechanism of promoting Treg features for immune regulation [11].
A close relationship exists between Th17 and Treg. Treg differentiating into Th17 cells in presence of IL-6 and into IL-17-producing FoxP3 + T cells in the synovia of active autoimmune arthritis [12,13] encourage the ongoing debate on plasticity of these two cell groups. Th17-like Treg and Treg expressing CD161, a characteristic marker for Th17/Th1 switching cell lines, seem to maintain suppressive function unless Treg start to produce IL-17 through activation by a pro-inflammatory cytokine environment [14][15][16].
Serum and synovial IL-17 levels were elevated in JIA patients and IL-17 significantly contributed to the proinflammatory conditions in specific JIA subtypes [17]. Secukinumab is a human monoclonal antibody directed against IL-17A. Similar to the positive effect in psoriatic arthritis and ankylosing arthritis in adults, the blockade of IL-17 may be useful especially in the JIA subtypes with high IL-17 levels like psoriatic and enthesitis-related arthritis [18]. So far, an approved pediatric indication for Secukinumab is psoriasis, phase III studies for enthesitisassociated and psoriatic JIA are ongoing.
To this date, only frequency and phenotype of Treg and Th17 have been studied in JIA. It remains unclear whether the targeted suppression of IL-17 is able to positively influence the regulatory function of Treg to control pro-inflammatory effectors in JIA and whether FoxP3 is up-regulated in this process. Thus, this study aimed to assess the influence of a Th17-stimulating cytokine environment and of inhibition of IL-17A not only on the plasticity of the Treg phenotype but also on the suppressive function of Treg derived from JIA patients as a potential add-on effect of IL-17A-targeted therapy.

Methods
Blood samples of 14 patients diagnosed with extended (n = 9) or persisting oligoarticular (n = 1) and seronegative polyarticular (n = 4) JIA according to ILAR-classification [19] were collected at the German Center for Pediatric and Adolescent Rheumatology, Garmisch-Partenkirchen. Due to known differences in pathophysiology [20], other JIA subtypes like enthesitis-associated, psoriatic or systemic JIA were excluded. Blood samples of 10 immunologically healthy controls (HC) were obtained at the University Hospital Wuerzburg. Exclusion criteria were malignancies, hematological diseases, monogenetic syndromes, severe infection requiring medical support in the past 3 months prior to inclusion, severe allergies and status post transplantation. No autoimmune disorders were reported in HC or first line relatives. The study was performed as an explorative prospective controlled cohort study according to the principles of the Declaration of Helsinki 2013 and was approved by the local ethics committee at the University Hospital Wuerzburg (protocol No. 239/10).
First, a characterization of CD4 + T helper (Th) cells was performed. Peripheral blood mononuclear cells (PBMC) were obtained from venous blood samples with density gradient centrifugation with FicoLite-H (Linaris blue GmbH, Wertheim, Germany) according to the standard laboratory protocol, stored until use in liquid nitrogen after preparation with 10% dimethyl sulfoxide (DMSO, Carl Roth GmbH, Karlsruhe, Germany) and analyzed with flow cytometry by staining after four hours of stimulation with Phorbol 12-myristate 13-acetate (PMA), Ionomycin and Brefeldin A (Sigma-Aldrich, St. Louis, USA) for surface molecules (CXCR3, CCR6, CD127, CD161, CD25, CD27, CD4, and zombie dye to exclude dead cells, all purchased from BioLegend, San Diego, USA) and with intracellular markers of Th17 and Treg cells (FoxP3, RORγt, IL-17, BD Biosciences, Franklin Lakes, USA and BioLegend) as described previously [21]. Differences between JIA and HC were assessed with BD FACS Diva Software Version 6.1.3.
To analyze the influence of different cytokines on Treg phenotype a magnetic-assisted isolation of CD4 + CD25 + CD127 − cells defined as isolated Treg was performed with Miltenyi Biotec MACS Treg Kit II (Bergisch-Gladbach, Germany) as described earlier [21].
The influence of the various stimuli on the suppressive function of Treg on autologous PBMC was assessed utilizing dilution of Carboxyfluorescein-succinimidyl-ester (CFSE, BioLegend) for detection of proliferated PBMC in suppression assays as described previously [21]. PBMC and Treg were co-cultured at equal amounts of cells in Th17-inducing cytokine conditions or under blockade of IL-17A as well as in negative and positive control conditions as described above. Inhibition of PBMC by Treg in % was determined as 1 minus the fraction of proliferation of PBMC in 1:1 co-culture with Treg and the proliferation of PBMC without Treg multiplied by 100.
After testing for distribution (Shapiro-Wilks-test), Mann-Whitney-U-test was applied for non-parametric, continuous independent variables (statistics software SPSS version 24.0, IBM statistics, Chicago, USA). A p-value less than 0.05 was considered statistically significant. Correction of type I error due to multiple comparisons was done with Bonferroni correction for each experiment, respectively.

Results
Demographics and clinical parameters of the study group are shown in Table 1. Thirteen patients (92.9%) were on medication and presented to be in laboratory remission according to C-reactive protein, erythrocyte sedimentation rate, total leucocyte, lymphocyte and thrombocyte counts (all within normal age-range) with clinically active joints in only four patients.
After 5 days of cultivation of isolated CD4 + CD25 + CD127 − Treg, an increase of FoxP3 + helper T cells (median 67.9%) in Th17 environment was found compared to HC (54.2%) (p = 0.026) and compared to unstimulated control (56.5%) (p = 0.015) (Fig. 1). In contrast to IL-17A blockade by the research-use-only antibody, where still a significant difference in FoxP3expression was seen between JIA and HC (p = 0.004), in vitro treatment with Secukinumab adjusted FoxP3expression of JIA-derived Treg to normal levels found in HC (56.7% vs. 49.3%; p = 0.247). There was no significant difference in the proportions of IL-17 + in FoxP3 + CD4 + cells after different Th17-stimulatory and IL-17A-inhibitory treatments in cultivated Treg (data not shown).
An analysis of the suppressive effect of isolated Treg on co-cultured autologous PBMC in different cytokine environment was performed to determine the influence of the stimuli on the functional capacity of the isolated Treg. Inhibition of PBMC proliferation by Treg was mitigated compared to the positive control when cultured with additional Th17-inducing cytokines both in JIA (52.5% vs. 21.5%; p = 0.032) and HC (60.5% vs. 32.5%; p = 0.005). IL-17A blockade with the research-useonly antibody as well as in vitro inhibition of IL-17A by Secukinumab resulted in steady and almost equal inhibition of PBMC proliferation compared to positive control in both JIA (50.0% vs. 52.5%; p = 1.00) and HC (59.1% vs. 60.5%; p = 0.534) (Fig. 2).

Discussion
Our findings demonstrate significantly elevated proportions of Th17 cells and Th17-like Treg in peripheral CD4 + helper T cells derived from JIA patients compared to HC. A shift towards Th17 and Th17-like Treg has been described by others [3,12,17,18]. The elevated proportions of IL-17 producing FoxP3 + CD4 + cells found in our cohort, may reflect either a specialization of Treg on Th17 cells in JIA as discussed by Campbell et al. for autoimmune diseases in general [8] or echo an increased plasticity of Treg towards the proinflammatory Th17 type [14]. Additionally, IL-17 producing cells derived from former Treg are discussed to be especially pathogenic in arthritic environment, e.g. they seem to be even more osteoclastogenic than for example Th17 cells from naïve CD4 + cells are [12]. Opposed to these findings of elevated Th17 and Th17like Treg we discovered lower proportions of RORγt in JIA patients compared to HC. This might be due to a reciprocal relation to the transcription factor FoxP3 which is known to suppress the expression of RORγt [4]. Furthermore, CCR6 and CD161 were introduced as markers of Th17/Th1 switching cells elevated in JIA compared to HC. Elevated Th17/Th1 switching cells have also been described earlier by Nistala et al. in inflamed joints of JIA patients [22].
Our in vitro results provide experimental evidence of plasticity of JIA-derived Treg with FoxP3-induction by Th17-stimulating cytokines mimicking in vivo inflammation. Whereas one study revealed the loss of FoxP3 in the presence of IL-6 [8], others demonstrated stable FoxP3 expression in Treg in an inflammatory environment [10] and furthermore, an increased FoxP3 expression in T cells by cell activation and by TGFβ [6]. The high sensitivity of JIA-derived Treg for Th17-inducing stimuli seen in higher level of FoxP3 + cells compared to HC may corroborate the supposed idea of an involvement of Th17-mediated pathways in the pathogenesis of many T cell-mediated autoimmune diseases [8,14], such as JIA.
In contrast to increased FoxP3 expression in JIA in isolated Treg after 5 days of incubation in cell culture media enriched with Th17-inducing stimuli, specific in vitro blockade of IL-17A resulted in adjustment of FoxP3expression to normal proportions as found in controls which might underline one mechanism of clinical effectiveness of IL-17 blockade in rheumatic diseases.
Specific IL-17A blockade with Secukinumab seems to be more efficient to allow adjustment to normal levels of FoxP3 expression than IL-17A blockade by the researchuse-only antibody. Although there is no literature yet, it

Table 1 Characteristics of the study population (A) and clinical data of Juvenile Idiopathic Arthritis patients (B)
Characteristics of the study population are shown in (A). Values are given in absolute numbers (percentages) or as mean ± SD (median; minimum to maximum).  might be explained by unknown mechanistic differences between the inhibitory function of Secukinumab and of the research-use-only antibody. Increased level of FoxP3 in the case of Th17-inducing stimuli may furthermore not be an expression of enrichment of naturally occurring Treg but more likely a hint of CD4 + T cell activation or induction of the induced Treg phenotype [11,23]. Although our experiments are based on peripherally circulating Treg cells, simulation of an inflammatory environment (e.g. as seen in inflamed joints) results in a significant reduction of inhibitory capacities of separated Treg on autologous PBMC. Our functional assays may support the finding that the phenotypically characterized FoxP3 + Treg are rather induced Treg which have at least partly lost or even not skilled their suppressive functions. Further on, an increased FoxP3-expression concomitant with mitigated inhibition may also be due to an increased resistance of effector cells towards Treg rather than to an impaired Treg function [16,24]. Such lower sensitivity of different effector cells towards Treg suppression was discussed in other experiments of our research group of lymphocytes derived from patients with scleroderma [25].
The limitations of this study include the high heterogeneity of JIA disease itself, of therapy and frequency of remittent disease and inflammatory activity. To overcome these problems, only oligo-and polyarticular subtypes and only JIA patients in laboratory remission or with very low clinical disease activity in four patients were included in the study. Enthesitis-associated, psoriatic and systemic JIA were not included due to the known differences in pathophysiology and presentation [20,26]. The highly standardized experimental protocol on separated Treg and culture in a pro-inflammatory and Table 2 Comparison of Th17-and regulatory T cells-specific markers in peripheral CD4 + helper T cells Peripheral blood mononuclear cells were assessed for Th17-and regulatory T cells (Treg)-characteristic markers by flow cytometry. Values of 5 healthy controls (HC) and 8 patients with Juvenile Idiopathic Arthritis (JIA) were compared with Mann-Whitney-U-test. Data are presented as percentages in mean ± standard deviation (median; minimum-maximum). After correction for multiple comparison with Bonferroni correction the results marked with * remained statistically significant. Elevated proportions of Th17/Th1 switching cells as well as CD4 + and CD4 + FoxP3 + cells producing IL-17 were assessed in JIA patients compared to HC  an anti-inflammatory environment allows to analyze the Treg function in-depth in a small sample of JIA patients while still keeping in mind the intrinsic heterogeneity of JIA subtypes. Possibly, findings would have been more jutting out using lymphocytes obtained during acute disease flare or from inflamed joints of therapy-naive recently diagnosed JIA patients. Furthermore it is important to mention that aging changes the cell subtypes of the immune system, therefore our study represents the examined adolescent group, not young children [27]. Additional experiments regarding epigenetic factors of FoxP3 stability as well as changes at the transcriptome level as performed in scleroderma [21,28] would have been interesting to assess but were not possible due to relatively low cell counts in volume-restricted blood samples in JIA patients.

Conclusion
In conclusion, our study supports findings of a shift towards Th17 and Th17-like Treg in JIA, underlining the importance of the Th17 pathway in the pathophysiology of JIA. Furthermore, our experiments show an increased FoxP3-expression in Treg by Th17-inducing cytokines with concomitant mitigated suppressive function. Whereas JIA Treg showed higher FoxP3-expression suggesting stronger cell activation by inflammatory stimuli, specific IL-17A blockade with Secukinumab lead to adjusted FoxP3 levels and suppressive function as found in controls. This may display a potential addon benefit of specific IL-17A blockade in JIA patients and therefore may help to provide supporting experimental data for the clinical use of IL-17A inhibition in JIA patients.  Values are shown in percentage (median and interquartile ranges). As stimuli were used: unstimulated = none; anti-CD3/CD28 as positive control; Th17 = anti-CD3/CD28 + IL-1β + IL-6 + IL-23 + TGFβ; anti-IL17A = anti-CD3/CD28 + laboratory IL-17A-antibody; Secukinumab = anti-CD3/28 + Secukinumab. N = 7 for each stimulus in healthy controls (HC), except for anti-CD3/anti-CD28 (n = 6) and n = 5 for each stimulus in Juvenile Idiopathic Arthritis (JIA). For stimuli comparison all stimuli were compared with anti-CD3/anti-CD28, which served as positive control. Groups as well as stimuli were compared for differences with Mann-Whitney-U-test. Mitigated inhibitory function was seen in Th17 environment in both groups. Difference between positive control and Th17-inducing cytokine environment in HC was significant (p = 0.005, marked with *) after Bonferroni correction was applied. Difference between negative control (= unstimulated) and positive control in HC was statistically significant as well (p = 0.001, not marked)


Inhibition of growth, biofilm formation, virulence, and surface attachment of Agrobacterium tumefaciens by cinnamaldehyde derivatives

Agrobacterium tumefaciens, a soil-borne, saprophytic plant pathogen that colonizes plant surfaces and induces tumors in a wide range of dicotyledonous plants by transferring and expressing its T-DNA genes. The limited availabilities and efficacies of current treatments necessitate the exploration of new anti-Agrobacterium agents. We examined the effects of trans-cinnamaldehyde (t-CNMA) and its derivatives on the cell surface hydrophobicity, exopolysaccharide and exo-protease production, swimming motility on agar, and biofilm forming ability of A. tumefaciens. Based on initial biofilm inhibition results and minimum inhibitory concentration (MIC) data, 4-nitro, 4-chloro, and 4-fluoro CNMAs were further tested. 4-Nitro, 4-chloro, and 4-fluoro CNMA at ≥150 μg/ml significantly inhibited biofilm formation by 94–99%. Similarly, biofilm formation on polystyrene or nylon was substantially reduced by 4-nitro and 4-chloro CNMAs as determined by optical microscopy and scanning electron microscopy (SEM) and 3-D spectrum plots. 4-Nitro and 4-chloro CNMAs induced cell shortening and concentration- and time-dependently reduced cell growth. Virulence factors were significantly and dose-dependently suppressed by 4-nitro and 4-chloro CNMAs (P ≤ 0.05). Gene expressional changes were greater after 4-nitro CNMA than t-CNMA treatment, as determined by qRT-PCR. Furthermore, some genes essential for biofilm formation, motility, and virulence genes significantly downregulated by 4-nitro CNMA. Seed germination of Raphanus sativus was not hindered by 4-nitro or 4-fluoro CNMA at concentrations ≤200 μg/ml, but root surface biofilm formation was severely inhibited. This study is the first to report the anti-Agrobacterium biofilm and anti-virulence effects of 4-nitro, 4-chloro, and 4-fluoro CNMAs and t-CNMA and indicates that they should be considered starting points for the development of anti-Agrobacterium agents.

Introduction
Agrobacterium tumefaciens is a fatal plant pathogenic bacterium responsible for crown-gall disease and contains a Tiplasmid that is inserted into the plant genome via horizontal gene transfer (Liu and Nester, 2006). This exclusive feature of A. tumefaciens has been well researched and utilized for genetic transformations of plants under laboratory conditions (Nguyen et al., 2021). However, in natural environments, pathogenic agrobacteria may infect a range of important crop plants based on their biovars; (i) A. tumefaciens species complex (biovar I), (ii) Agrobacterium rhizogenes (biovar II), and (iii) Agrobacterium vitis (biovar III) (Slater et al., 2009). Of these, A. tumefaciens is predominantly found living a saprophytic lifestyle in different environments, including the rhizosphere where it thrives, forms biofilms (Heindl et al., 2014), and may infect a broad range of dicotyledonous plant species (>600), induce gall formation, and cause huge crop losses (>5% of economically important crops globally) (Moens, 2009). A. tumefaciens senses chemical signals (rhizospheric signal molecules), such as sugars, organic acids, and amino acids by chemotaxis, and enters host tissues at surface wounds (Liu and Nester, 2006). Furthermore, on receiving signals from plants, Agrobacterium increases the expressions of its virulence genes. The two-component VirA/VirG regulatory system activates virulence genes and assists transfer of Ti-plasmid to host plants (Nabi et al., 2022). Virulent A. tumefaciens transfers and integrates its T-DNA fragment from a Ti-plasmid into the host genome and its subsequent expression increases the production of opines and plant hormones like cytokinin and indole-3-acetic acid (Dessaux and Faure, 2018;Nabi et al., 2022), which enhance the plant growth and induce tumor formation. Opines are utilized by Agrobacterium as nutrients and activate quorum sensing (QS) signaling, which further enhances A. tumefaciens virulence and opine metabolism (Faure and Lang, 2014).
For disease to occur, A. tumefaciens must first physically attach to the host surface, which may occur in a stepwise manner, as follows: (i) initial surface contact by motile flagella, (ii) establishment of transient reversible attachment facilitated by protein adhesins and a range of pili (conjugative and Ctp) (Matthysse, 2014), and (iii) irreversible attachment by bacterial exopolysaccharides (EPS) after biofilm establishment (Thompson et al., 2018). A. tumefaciens can colonize and form biofilms on various abiotic surfaces, plant roots, and wounds, which it reaches by swimming using six flagella located around a single pole (Merritt et al., 2007) and then attaches firmly to cellulose fibrils. Bacterial EPS secretion, pili activity, and biofilm formation vary among soil-borne pathogenic bacteria but are required when pathogens transit to the biofilm mode from planktonic (Muhammad et al., 2020). Generally, antibiotics, copper bactericides, or fosetyl-aluminum are used to control plant pathogenic bacteria, but these measures are less effective at controlling A. tumefaciens infections , not cost-effective, and not readily available. Agrocin 84 (a biopesticide) produced by genetically modified (GM) Agrobacterium radiobacter (non-pathogenic) strains K84 and K1026, which competitively colonized the roots of several crops, were reported to inhibit the production of leucyl-tRNA synthetase in A. tumefaciens (McCardell and Pootjes, 1976;Kerr and Bullard, 2020). However, its effects on non-targeted useful rhizospheric organisms have not been assessed, and its interactions with agrobacterial species complex (biovar I-III) are unknown. Furthermore, field applications of GM organisms are prohibited in some countries (Kahla et al., 2017). Thus, other novel approaches are needed to prevent A. tumefaciens biofilm formation and virulence and control crown gall disease.
Plant-derived bioactive compounds offer a potential source of anti-Agrobacterium molecules, and cinnamaldehydes derived from the bark of ∼250 species belonging to the genus Cinnamomum are of particular interest (Shreaz et al., 2016). The cinnamaldehyde obtained from essential oils has been categorized as generally regarded as safe (GRAS) by the U.S. Food and Drug Administration (FDA) and has been approved for used in foods (Wei et al., 2011) and given status "A" by the Council of Europe for use in food (Friedman, 2017). Due to its characteristic aroma, color, and taste, transcinnamaldehyde (t-CNMA) is used medically and as a flavoring agent (Chun et al., 2013). Furthermore, t-CNMA has been reported to have anti-QS (Zhang et al., 2018), antibiofilm , and antibacterial effects (Yossa et al., 2014) against several food and clinical pathogens including Erwinia carotovora (Zhang et al., 2018), Pseudomonas fluorescens (Zhang et al., 2018), Campylobacter spp. , Escherichia coli O157:H7 (Yossa et al., 2014), Salmonella (Yossa et al., 2014), Staphylococcus aureus (Ferro et al., 2016), and the fungus Candida albicans (Chen et al., 2019). Moreover, cinnamaldehydes also have antioxidant, anti-inflammatory, anticancer, and anti-diabetic activities (Rao and Gan, 2014). Nevertheless, the antivirulence and antibiofilm effects of t-CNMA and its derivatives on A. tumefaciens have not been investigated. We hypothesized that t-CNMA and its derivatives might inhibit biofilm formation by A. tumefaciens on abiotic and biotic surfaces, and thus, we investigated the antibacterial and antibiofilm effects of t-CNMA and ten of its derivatives.

Organism and growth conditions
Agrobacterium tumefaciens GV2260 was maintained at 30 • C on Luria Bertani (LB) agar plates, and for long-term preservation, glycerol (20% v/v) culture stocks in LB were stored at −80 • C. For working cultures, two independent colonies from LB agar plates were inoculated in LB broth and incubated at 250 rpm for 24 h at 30 • C. At least two independent cultures replicated into three (n = 2 × 3 = 6) were used for experiments.

Antibiofilm screening and minimum inhibitory concentration determinations
For antibiofilm screening, colonies of A. tumefaciens grown for 24 h in LB broth were diluted with fresh LB at 1:50, and t-CNMA or its derivatives at 100 or 200 µg/ml in LB were added. Bacterial cells incubated with LB only were considered non-treated controls. Aliquots (300 µl) of these cultures were added to wells of 96-well microtiter plates and incubated for 48 h at 30 • C. Then A. tumefaciens biofilm formation was checked using a crystal violet assay (Ramey and Parsek, 2006), with modifications; wells were washed three times with sterile distilled water kept at room temperature, plates were air-dried, and 300 µl of 0.1% crystal violet was added to each well. After incubation for 20 min at room temperature, wells were rinsed three times with sterile distilled water, and 300 µl of 95% ethanol was added. After shaking microtiter plates for 1 min in a plate reader, biofilm absorbance was recorded at 570 nm. To determine minimum inhibitory concentrations (MICs), A. tumefaciens cells were treated with 0-400 µg/ml of t-CNMA or its derivatives in LB diluted at 1:100, and then 300 µl aliquots were incubated in a microtiter plate for 24 h at 30 • C. Cell growths were determined by measuring optical density at 620 nm.

Estimation of Agrobacterium tumefaciens biofilm production by crystal violet assay: Quantification and microscopy
Cells of strain GV2260 from 24 h cultures were diluted with LB at 1: 50 and 25, 50, 75, 100, 150, 200, or 400 µg/ml of t-CNMA, 4-nitrocinnamaldehyde (4-nitro CNMA), 4-chlorocinnamaldehyde (4-chloro CNMA), or 4fluorocinnamaldehyde (4-fluoro CNMA) were added. Crystal violet biofilm assays were performed as described in section "Antibiofilm screening and minimum inhibitory concentration determinations" above. A total of six wells and two independent bacterial cultures were used for each test concentration. The experiment was terminated after incubation for 48 h at 30 • C under static conditions.
For microscopic observation, biofilm formation by A. tumefaciens was also challenged in 6-well tissue culture plates using t-CNMA, 4-nitro, 4-chloro, or 4-fluoro CNMA. Briefly, 3 ml of bacterial suspensions prepared in LB broth (1:50 culture broth ratio) were mixed with t-CNMA or its derivatives (50-200 µg/ml) and added to wells. After incubation for 48 h at 30 • C under static conditions, the media containing planktonic cells was carefully removed, and biofilms were rinsed carefully three times with PBS in the wells. Biofilms were then stained with crystal violet (0.1%) for 20 min at room temperature, rinsed with distilled water, and visualized using the iRiS Digital Cell imaging system (Logos Biosystems, Annandale, VA, USA). Micrographs were captured, and color mesh plots were created using ImageJ software.

Effect of trans-cinnamaldehyde and derivatives on biofilm formation on membranes
Agrobacterium tumefaciens was allowed to form biofilm on nylon membrane surfaces in 96-well plates (Lee et al., 2011). t-CNMA, 4-nitro CNMA, 4-chloro CNMA, or 4-fluoro CNMA (200 µg/ml) were added to bacterial cultures in LB (1:50), and small autoclaved pieces of nylon membrane were added to wells. Plates were incubated for 48 h at 30 • C. Membranes were then removed, rinsed with sterile PBS, fixed in a mixture of 2.5% glutaraldehyde and 2% formaldehyde solution in distilled water, left for 30 min at room temperature, and then kept at 4 • C overnight (Ahmed et al., 2021). Samples were then dehydrated using an ethanol gradient (30,50,70,90, and 100% for 10 min each), critical point dried, coated with Au or Pt, and visualized by FE-SEM (model S-4200, Hitachi, Tokyo, Japan) at 15 kV. The length of biofilm cells attached to nylon surface were determined by measuring the length of at least 50 cells per test concentration using ImageJ software. A scale was set with the "Analyze" tool of ImageJ and then length of each cell (n = 50) was determined in micrometers (µm).
Virulence factor production by Agrobacterium tumefaciens

Swimming motility
The swimming motility of A. tumefaciens GV2260 was assessed using peptone-agar (1% peptone, 0.25% agar, and 0.5% NaCl) containing t-CNMA or its derivatives at 25-200 µg/ml. The bacterial inoculum (1 µl) from overnight grown culture was placed at the center of peptone-agar plates and allowed to stand for 72 h at 30 • C (Ahmed et al., 2022). Swim diameters were recorded at 48 and 72 h to check cell migration through agar. Data from three replicates were averaged.

Measurement of cell surface hydrophobicity
Agrobacterium tumefaciens was exposed to t-CNMA or its derivatives at 25, 50, 75, 100, 150, 200, or 400 µg/ml in 1 ml LB at a culture: medium ratio of 1:100 in Eppendorf tubes and incubated at 250 rpm for 24 h at 30 • C. Microfuge tubes were then centrifuged at 10,000 rpm for 10 min, and cell pellets were mixed with PBS, washed three times, and resuspended in PBS (1 ml). Optical density (OD) values of suspensions were read at 600 nm and designated A 0 . The method described earlier for bacterial adhesion to hydrocarbons (BATH) was followed (Rosenberg, 1984) with modifications. Hexadecane was then added to cell suspensions and vigorously vortexed for 1 min and then left for 30 min at room temperature to allow phase separation. Similarly, a blank (1 ml PBS only) was also processed. Aqueous phase OD (600 nm) values were designated A i. Percent hydrophobicity were calculated using the formula:

Assessment of extracellular protease production
Agrobacterium tumefaciens culture mixed with LB at 1:100 was exposed to t-CNMA compounds at 25-400 µg/ml in LB at 250 rpm for 24 h at 30 • C. Samples were then spun at 10,000 rpm for 10 min, and supernatants were collected. Supernatants (100 µl) were mixed with an equal volume of azocasein and incubated for 30 min at 37 • C when 600 µl of tricarboxylic acid (10%) was added to stop proteolysis. These mixtures were then kept for 30 min at −20 • C. After centrifugation at 10,000 rpm for 10 min, 700 µl of the resulting supernatants was added to 700 µl of NaOH, and absorbances were recorded at 440 nm (Sethupathy et al., 2020).

Determination of exopolysaccharides production
Agrobacterium tumefaciens was grown with or without t-CNMA derivatives at 25, 50, 75, 100, 150, 200, or 400 µg/ml in LB in 1.5 ml microfuge tubes at 250 rpm for 24 h at 30 • C. Tubes were centrifuged at 10,000 rpm for 10 min. Supernatants were added with chilled ethanol at a ratio of 1:3 and left undisturbed at 4 • C for overnight. EPS precipitates were collected by centrifugation (10,000 rpm for 5 min.) and solubilized in 200 µl of water. A phenol/sulfuric acid mixture (prepared at a ratio of 1:5) was then added to 200 µl of EPS samples, incubated for 30 min. at room temperature, and left at room temperature for 20 min. Absorbances were measured at 490 nm (Ali et al., 2016).

Time-dependent growth inhibition assay
Agrobacterium tumefaciens was grown in LB diluted at 1:100 for 24 h then treated with t-CNMA or its derivatives at different concentrations. These suspensions (300 µl) were then added to the wells of a 96-well plate. Culture growths were monitored every 2 h at 620 nm for 24 h (Ahmed et al., 2019b). The results of two independent cultures in six wells per concentration were averaged and plotted as a function of incubation time and concentration.

Impact of trans-cinnamaldehyde derivatives on CFU count and percent cell survival
Agrobacterium tumefaciens grown for 24 h in LB was diluted at 1:100 with 25-400 µg/ml of t-CNMA, 4-nitro CNMA, 4chloro CNMA, or 4-fluoro CNMA and incubated at 250 rpm for 24 h at 30 • C. Aliquots (100 µl) of appropriate dilutions were then plated on LB agar and incubated for 48 h at 30 • C. Colonies were counted, and CFU/ml values were calculated and converted to a log scale: Survival percentages of A. tumefaciens were also calculated with respect to non-treated controls.
Biofilm formation by trans-cinnamaldehyde derivatives-challenged Agrobacterium tumefaciens on root surfaces Seeds of Raphanus sativus were germinated on 0.86 g/L MS medium supplemented with 0.7% agar, and after germination,

qRT-PCR analysis of Agrobacterium tumefaciens genes
To assess the expressional changes induced by t-CNMA and 4-nitro CNMA, qRT-PCR was used to analyze the expressions of motility (flgE and motA), biofilm (celA, cheA, and phoB), virulence (virE2, chvE, virE0, and virG), stress related response (clpB, dnaK, gsp, marR, soxR, and hspAT2), and efflux pump (emrA, norM, ifeA, and ifeR) genes. A. tumefaciens culture in 25 ml LB with an OD 600 nm of 1.0 was incubated with 100 µg/ml of t-CNMA or 4-nitro CNMA at 250 rpm for 8 h at 30 • C. An RNase inhibitor (700 µl; RNAlater, Ambion, TX, USA) was added and gently agitated on ice. Centrifugation of untreated and treated cultures was performed at 13,000 rpm for 10 min at 4 • C. The RNA was extracted using the Qiagen RNeasy mini kit (Valencia, CA, USA) and concentrations were determined using a nanodrop spectrophotometer (model: Cytiva NanoVue Plus Spectrophotometer, Fisher Scientific, England, UK). Primer sequences of tested genes are provided in Supplementary  Table 1. qRT-PCR was conducted using SYBR green master mix and an ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using two independent cultures .

Evaluation of seed germination
The impact of t-CNMA and its derivatives on white radish (R. sativus) seed germination was examined (Ahmed et al., 2019a). In brief, R. sativus seeds were soaked in distilled water for 6 h, thoroughly rinsed with distilled water and then ethanol (95%), and surface sterilized using sodium hypochlorite (3% for 10 min). t-CNMA its derivatives at 25-400 µg/ml were added to soft agar (0.7% agar) containing 0.86 g/L Murashige and Skoog (MS) medium. After washing with autoclaved distilled water, 10 seeds/plate/test were placed and incubated at 25 • C for 4 days. The seeds showing evidence of germination were counted.

Data analyses
All experiments were performed with two independent bacterial cultures. Data are expressed as means ± standard deviations (SD), and significances of differences were determined using the two-tailed t-test. Statistical significance was accepted for P-values ≤ 0.05 unless otherwise stated. Graphs were prepared using Sigma Plot Ver. 14.0.

Determination of minimum inhibitory concentrations of cinnamaldehydes against Agrobacterium tumefaciens
The effects of t-CNMA and 10 of its derivatives on A. tumefaciens were investigated. As shown in Table 1, MICs were variable. We selected three derivatives, viz. 4-nitro CNMA, 4-fluoro CNMA, and 4-chloro CNMA based on their MICs, which were 100, 200, and 200 µg/ml, respectively, and their biofilm inhibitory effects at 100 or 200 µg/ml ( Table 1). t-CNMA (MIC 400 µg/ml) was used as the control.

Impact of trans-cinnamaldehyde derivatives on exopolysaccharides production and cell surface hydrophobicity
The QS regulates virulence factors considered responsible for A. tumefaciens biofilm formation (Faure and Lang, 2014). Of these, cell surface hydrophobicity and extracellular polymeric substances are critical for bacterial adhesion and successful biofilm formation. Non-treated cells of A. tumefaciens produced significant amounts of EPS; however, treatments with t-CNMA Inhibition of A. tumefaciens biofilm formation on flat polystyrene in 6-well plates as determined by crystal violet staining (A) and optical microscopy at 20× magnification (B) by t-CNMA, 4-nitro CNMA, 4-chloro CNMA, and 4-fluoro CNMA in the concentration range (50-400 µg/ml). or its derivatives decreased EPS production at relatively hightest concentrations (Figures 4A-D). t-CNMA did not reduce the EPS secretion below 150 µg/ml but decreased it significantly by >40% at concentrations ≥200 µg/ml ( Figure 4A). 4-Nitro CNMA at 75 µg/ml caused a similar reduction (40%) and at 200 µg/ml decreased EPS production by 69% (Figure 4B). 4-Chloro CNMA caused a significant reduction at 100 µg/ml followed by a concentration-dependent decrease till 400 µg/ml ( Figure 4C). The reduction in EPS production by 4-fluoro CNMA (Figure 4D) was similar to that of t-CNMA. Cell surface hydrophobicity (% CSH) is essential for bacterial attachment to surfaces (Tribedi and Sil, 2014) and was also found to be decreased by t-CNMA and its three derivatives in a concentration-dependent manner (Figures 4E-H). When we compared the inhibitory effects on % CSH at a concentration of 100 µg/ml, 4-nitro CNMA ( Figure 4F) and 4-chloro CNMA ( Figure 4G) had the greatest effects and reduced CSH by 79% (P ≤ 0.05) and 82% (P ≤ 0.05), respectively, versus non-treated controls. At 150-400 µg/ml, %CSH was zero for t-CNMA ( Figure 4E) and 4-nitro CNMA ( Figure 4F). As was observed in the EPS assay, 4-fluoro CNMA had least reduction (45% at 200 µg/ml) (Figure 4H).

Reduced biofilm formation on Raphanus sativus roots
Agrobacterium tumefaciens formed biofilms on R. sativus roots after incubation for 48 h under non-treated and optimized growth conditions (Figures 7A-C). The biofilms produced were dense and showed multiple aggregates of cells embedded in an EPS-like substance ( Figure 7C). While observing root surfaces, biofilms were observed in multiple regions after t-CNMA treatment (Figures 7D-F). However, no biofilms were observed on roots after exposure to 4-nitro CNMA (Figures 7G-I), 4-chloro CNMA (Figures 7J-L), or 4-fluoro CNMA (Figures 7M-O), though a few cells were dispersed at some locations on root surfaces (Figures 7I,O). Furthermore, root surfaces in different zones, e.g., root tips, hairs, and meristematic and root elongation zones were undamaged after exposure to 200 µg/ml of the test compounds. SEM findings after 4-fluoro CNMA treatment (Figures 7M-O) differed from nylon membrane results (Figures 3J,K) since 4-fluoro CNMA did not eradicate biofilms on nylon membranes. On the other hand, results for t-CNMA, 4-nitro CNMA, and 4-chloro CNMA treatments of biofilms on nylon (Figure 3) and R. sativus root surfaces (Figure 7) were correlated.

Changes in gene expressions induced by trans-cinnamaldehyde and 4-nitro cinnamaldehyde
Biofilm, virulence, stress, motility, and efflux pump regulation genes of A. tumefaciens (OD 600 nm = 1.0) assessed after 8 h contact with test compounds in LB at 30 • C were Production of virulence factors and their inhibition by t-CNMA and its 4-nitro, 4-chloro, and 4-fluoro CNMA derivatives. Effects on EPS production (A-D), cell surface hydrophobicity (E-H), and extracellular protease production (I-L). "*" denotes a significant difference by the two-tailed t-test (P ≤ 0.05).
variably affected by t-CNMA and 4-nitro CNMA versus nontreated controls while 4-nitro CNMA more significantly affected the gene expression than t-CNMA (Figure 8). Genes subjected to qRT-PCR were selected based on their direct or indirect involvements with these functions. For example, the biofilm formation genes cheA, celA, and phoB encode for the twocomponent sensor kinase of the Che operon that regulates chemotaxis (Merritt et al., 2007), cellulose synthase required for cellulose production (Matthysse et al., 2005), and production of a regulatory protein for the two-component (PhoR-PhoB) system (Tomlinson et al., 2010), respectively. 4-Nitro CNMA significantly (P ≤ 0.05) downregulated celA, cheA, and phoB by 3. 3-, 3. 9-, and 3-fold, respectively, versus non-treated controls (Figure 8). Similarly, two flagellar motility genes flgE and motA, which encode for a flagellar hook protein and a constituent of the flagellar motor of A. tumefaciens, were downregulated by 11-and 2.5-fold, respectively, by 4-nitro CNMA (Merritt et al., 2007). Interestingly, t-CNMA only slightly reduced the expression of these genes (Figure 8). Among the virulence genes, virE2 encodes for virulence protein (virE2) that facilitates the import of T-DNA-protein complex in the host nucleus (Li et al., 2020). chvE encodes for a periplasmic-binding protein, which after interacting with the VirA/VirG regulatory system induces the expressions of vir genes (Hu X. et al., 2013). virE0 encodes for a regulator protein that may be directly involved in Agrobacterium-plant interactions (Yuan et al., 2008). virG encodes for a two-component response regulator protein (Yuan et al., 2008) and was significantly downregulated (Figure 8) by t-CNMA only. The expressions of all other virulence genes were either unchanged or non-significantly downregulated. Genes involved in multiple stress responses, namely, clpB, dnaK, gsp, marR, and hspAT2, reported in other studies (Rosen et al., 2001;Tsai et al., 2012;Rittiroongrad et al., 2016) were also tested. Results revealed that dnaK and clpB were downregulated by 2.7-and 28-fold, respectively, and soxR was upregulated (1.72) by 4-nitro CNMA (the expressions of other stress-related genes were slightly changed or unaffected). t-CNMA upregulated the gsp gene, which is associated with general stress, by 2.5-fold, and 4-nitro CNMA reduced the expression of a heat shock protein (hspAT2). Also, some efflux pump genes, namely, emrA, norM, ifeA, and ifeR, were included (Palumbo et al., 1998;Nuonming et al., 2018;Khemthong et al., 2019), but the only effect observed was that 4-nitro CNMA upregulated emrA by 3.7-fold (Figure 8).

Discussion
We report the biofilm inhibiting characteristics of t-CNMA and ten derivatives, which were selected because of their dissimilar functional moieties on the aromatic ring or side chain of t-CNMA. A few studies on CNMA derivatives have reported the antityrosinase effects of αsubstituted derivatives such as α-methylcinnamaldehyde, α-chlorocinnamaldehyde, and α-bromocinnamaldehyde (Cui et al., 2015). Similarly, fungal growth was inhibited by 2-bromo and 2-chlorocinnamaldehyde (Badawy and Rabea, 2013). Among the ten derivatives investigated in the present study, 4-nitro CNMA, 4-chloro CNMA, and 4-fluoro CNMA were subjected to further testing because they exhibited potent antibiofilm effects and low MICs. Badawy and Rabea (2013) reported that chitosan-based derivatives had antifungal activity against seven fungal species. The same authors investigated the effects of chitosan-based derivatives, such as N-(α-methylcinnamyl) chitosan and N-(o-methoxycinnamyl) chitosan, on A. tumefaciens but reported very high MICs of 1,275 and 1,925 µg/ml, respectively. In the present study, the MICs of 4-nitro CNMA, 4-fluoro CNMA, and 4-chloro CNMA were 100, 200, and 200 µg/ml, respectively. Zhu et al. (2009) previously reported t-CNMA thiosemicarbazone had an A. tumefaciens MIC of 100 µg/ml with no report on antibiofilm potential and gene expressional changes. Biofilms provide microorganisms on abiotic and biotic surfaces with well-structured protective sheaths impermeable to drugs and antibacterial agents (Sharma et al., 2019). Thus, microorganisms in biofilms are more virulent than planktonic cells, and novel solutions are required to address this challenge (Ying et al., 2019). A. tumefaciens adheres to surfaces using its molecular appendages (Thompson et al., 2018) and subsequently forms reversible or irreversible attachments (Heindl et al., 2014) with abiotic (Figures 2, 3) or biotic surfaces (Figure 7). After establishing contact with a surface, A. tumefaciens releases EPS to make this contact reversible Frontiers in Microbiology 11 frontiersin.org and initiates microcolony formation (Heindl et al., 2014).
In the present study, 4-nitro CNMA and 4-chloro CNMA significantly inhibited biofilm formation by A. tumefaciens on polystyrene and nylon (Figures 2, 3F-I) and plant root surfaces (Figures 7G-L), as determined by light microscopy and SEM. Similar reductions in bacterial aggregation and microcolony formation by P. fluorescens were observed by light microscopy after cinnamaldehyde exposure. In a previous study, SEM revealed a maximally disrupted biofilm architecture of P. fluorescens at 0.1 µl/ml t-CNMA (Li et al., 2018). We observed gaps and poor volumes of A. tumefaciens biofilms (Figure 3) after treatments with 4-nitro CNMA (150-200 µg/ml), 4-chloro CNMA (200 µg/ml), or 4-fluoro CNMA (400 µg/ml) but not after treatment with t-CNMA. Li et al. (2018) reported that t-CNMA induced fissures in P. fluorescens biofilms, and Kim et al. (2015) observed cinnamon bark oil and t-CNMA at 0.01% v/v reduced enterohemorrhagic E. coli (EHEC) fimbriae formation, which is required for biofilm maturation, and suggested that reduced EHEC fimbriae production by t-CNMA on nylon membranes was largely responsible for biofilm inhibition. Our observations of reductions in A. tumefaciens biofilm formation at ≥150 µg/ml by t-CNMA, 4-nitro CNMA, and 4-chloro CNMA (Figure 1) suggest that -NO 2 functional group at the fourth position on the aromatic ring is more detrimental to biofilm formation than -Cl, and that -F is less effective than -NO 2 or -Cl. When we compared the gene expressional changes induced by 4-nitro CNMA and t-CNMA, 4-nitro CNMA was found to have substantially more potent effects (Figure 8). Furthermore, our findings regarding the effects of t-CNMA concur with those of Budri et al. (2015) who found that at 106 µg/ml t-CNMA reduced MRSA biofilm formation on stainless steel and polystyrene by 45 and 70%, respectively. Similarly, Albano et al. (2019) found t-CNMA at 300 µg/ml reduced biofilm formation by Staphylococcus epidermidis by 89%.
Frontiers in Microbiology 13 frontiersin.org Gene expressional changes in A. tumefaciens induced by 100 µg/ml t-CNMA or 4-nitro CNMA after treatment in LB broth at 250 rpm for 8 h at 30 • C. Bars are averages of four reactions performed using two independent cultures. 16S rRNA was used as the housekeeping gene. "*" represents a significant difference (P ≤ 0.05) between non-treated and treated cells. Fold changes in gene expression were calculated using the 2 − Ct method.
Biofilm inhibition by t-CNMA might be related to disruption of the QS regulatory system, as has been reported for P. fluorescens (Li et al., 2018) and E. coli (Niu et al., 2006), and this inhibition may be due to the downregulations of curli genes (csgA and csgB) in EHEC  and biofilmrelated adhesion genes (icaA and sarA) in Staphylococcus spp. (Jia et al., 2011). QS signaling controls EPS secretion, motility, protease production, and cell surface hydrophobicity (Li et al., 2014;Tan et al., 2014;Mizan et al., 2016;Pena et al., 2019), and in the present study, these virulence attributes of A. tumefaciens were remarkably and concentration-dependently inhibited by t-CNMA and its derivatives (Figures 4, 5). For t-CNMA, these effects have been suggested to be associated with the aldehyde group (Kot et al., 2015) as also shown for swimming motility of E. coli at 2.17 mM t-CNMA (Niu and Gilbert, 2004). Furthermore, it has been suggested that the presence of halogen (-Cl or -F) or nitro (-NO 2 ) groups in t-CNMA increases its suppressive effects on bacterial virulence (Brackman et al., 2011;Nepali et al., 2018).
We also observed that 4-nitro CNMA, 4-chloro CNMA, and 4-fluoro CNMA reduced the lengths of A. tumefaciens cells (Figures 3G,I,K) as compared with non-treated ( Figure 3C) and t-CNMA ( Figure 3E) treated cells. However, we did not observe the t-CNMA-induced morphological distortions of E. coli and S. aureus cells reported by Shen et al. (2015). Furthermore, exposure to t-CNMA and the three derivatives had markedly impacted cell viability and growth (Figure 6), which could be associated with t-CNMA-induced reductions in intracellular pH (Oussalah et al., 2006), its interactions with membrane proteins (Mousavi et al., 2016), or its effects on cell membrane conductivity (He et al., 2019) or membrane lipid profiles (Wendakoon and Sakaguchi, 1995). In addition, the effects of -NO 2 , -Cl, and -F containing derivatives may have been influenced by the electronegativities of these groups (Shaikh et al., 2016;Doyle et al., 2019). t-CNMA has also been suggested to act as an ATPase inhibitor and inhibit enzymes involved in cytokine interactions (Shreaz et al., 2016). Furthermore, differences between the cellular uptakes of derivatives and their post-cellular uptake transformations may have modulated their effects.
No biofilm formation was observed on the root surfaces of R. sativus seedlings roots grown in the presence of 4-nitro CNMA, 4-chloro CNMA, or 4-fluoro CNMA (Figures 7G-O). A difference in biofilm volume on root surface and nylon surface was observed in non-treated groups where it was higher on nylon membranes possibly due to more firm attachment of cells on cellulose fibrils (Merritt et al., 2007;Heindl et al., 2014). The inhibition of A. tumefaciens biofilm formation on plant roots by CNMA derivatives has not been previously reported. However, biofilms of Pseudomonas putida KT2440 were reported to be dose-dependently inhibited by t-CNMA (Niu and Gilbert, 2004). A. tumefaciens utilizes adhesive pili, rhicadhesin, and chromosome-encoded factors in addition to universal forces like electrostatic and hydrophobic interactions and Van der Waals forces to attach to plant surfaces (Wheatley and Poole, 2018) and form biofilms. Our qRT-PCR data showed 4-nitro CNMA induced significant changes in the expressions of genes associated with motility (flgE and motA), biofilm formation (celA, phoB, and cheA), stress response (clpB, dnaK, and soxR), and efflux pump (emrA) (Figure 8) Mechanistic illustration of A. tumefaciens interaction with t-CNMA and its analogs specifically 4-nitro CNMA after screening based on MIC and biofilm inhibition percentages. A. tumefaciens was able to swim in agar by flagellar machinery, showed cell surface hydrophobicity, showed protease and EPS production, EPS and biofilm on three different surfaces nylon, polystyrene, and plant root (displayed beneath the biofilm). Selected derivatives inhibited these virulence factors and biofilm phenotype. Higher alterations in gene expressions were observed by 4-nitro CNMA than t-CNMA. Different color-coded gene boxes represent specific virulence phenotype (i.e. biofilm, motility, stress, and efflux), solid black bordered boxes show downregulation while blue bordered boxes show upregulation of genes. Arrows in different colors represent different group of genes and flat arrow ends stand for inhibition. cDNA, complimentary DNA; qPCR, quantitative polymerase chain reaction; MIC, minimum inhibitory concentration; EPS, exopolysaccharides. compared to t-CNMA. A. tumefaciens downregulated by 4nitro CNMA induced a series of events that ultimately result in inhibition of bacterial adherence on surfaces, EPS, motility, biofilm as depicted in Figure 9. For example, celA encodes for a regulatory protein of two-component sensor kinase required for chemotaxis, therefore its downregulation reduces the chemotactic response of A. tumefaciens towards rhizospheric chemicals and prevent adherence to plant tissues (Figure 9). Another biofilm gene cheA encodes for cellulose synthase and induces EPS production, therefore its inhibition brings down the EPS matrix of biofilm. The increased transcription of soxR (stress related gene), sensing stress due to 4-nitro CNMA presence induced biofilm formation, however, other genes regulating the biofilm formation directly or indirectly were downregulated. The stress-related clpB and dnaK (or Hsp70) were downregulated and this ATP-dependent protease production and correct folding of proteins were compromised respectively that is suggestive of the possible antibacterial mechanism of 4-nitro CNMA.
Our findings suggest that the presence of strong electron-withdrawing groups at the fourth position of t-CNMA disrupts the biofilm formation mechanism of A. tumefaciens. The Ca 2+ adhesion protein rhicadhesin has been proposed to play a role in A. tumefaciens attachment to plant surfaces (Swart et al., 1994), and it has been suggested that t-CNMA might similarly disrupt Ca 2+ homeostasis in Phytophthora capsici (Hu L. et al., 2013). After evaluating the effects of t-CNMA, 4-nitro CNMA, 4-chloro CNMA, and 4-fluoro CNMA on A. tumefaciens, we suggest that structure-based activity factors and the presence of a conjugated aldehyde contribute to antibiofilm effects of t-CNMA. Xie et al. (2017) assigned the antifungal activity of t-CNMA and α-methyl CNMA to -CHO and -CH 3 group at the ortho position of the aromatic ring. We found the presence of -NO 2 , -Cl, or -F at the para position had considerable effects on A. tumefaciens biofilms, and that 4-nitro CNMA had a greater suppressive effect on biofilm-associated genetic factors than t-CNMA (Figure 8).

Conclusion
In summary, we evaluated the antibiofilm and antivirulence effects of t-CNMA and 4-nitro CNMA, 4-chloro CNMA, and 4-fluoro CNMA on A. tumefaciens. t-CNMA significantly reduced swimming motility, cell surface hydrophobicity, EPS secretion, and exo-protease production; however, these effects were considerably greater for 4-nitro CNMA and 4-chloro CNMA. We suggest the greater effects of these two derivatives on biofilm formation and growth may have been due to the presence of (i) a conjugated aldehyde group and (ii) an electron-withdrawing group like -NO 2 at the para position. Also, qRT-PCR data showed 4-nitro CNMA downregulated the expressions of multiple biofilm formation associated genes, which shows CNMA derivatives target multiple processes and thus are unlikely to induce resistance in A. tumefaciens. Moreover, reductions in A. tumefaciens cell viability, growth, and root surface biofilm formation observed suggest that t-CNMA derivatives with -NO 2 , -Cl, or -F at position 4 on the aromatic ring provide an excellent starting point for the development of anti-Agrobacterium agents that effectively prevent crown gall disease.

Data availability statement
The original contributions presented in this study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.